Cell-free fetal DNA is present in the maternal circulation and by our methods amenable to robust recovery. Cell-free fetal DNA in plasma is even more abundant than DNA derived from intact cells in maternal blood. The biological (structural) form in which fetal DNA exists and the mechanisms underlying its variation in maternal plasma are unclear. Because great interest lies in the diagnostic utility of this fetal genetic material, structural and molecular characterization to facilitate improved isolation and/or enrichment of fetal DNA is warranted. Based on our preliminary data that apoptotic bodies and DNA are present in maternal plasma and that fetal specific sequences can be enriched 3-5 fold from flow sorted acridine orange stained plasma fractions containing these vesicles, we hypothesize that fetal DNA circulates predominantly in the form of apoptotic bodies. The presumptive cell type is placental villi (i.e. trophoblasts). We further hypothesize that fetal and maternal DNA differ in their molecular properties. In this revised proposal, our project aims are: 1) to determine the structural nature of circulating fetal DNA in plasma from pregnant women. Visual, morphologic and DNA-staining will be used to identify apoptotic bodies and nucleosomes in maternal plasma, and for subsequent enrichment using either flow cytometry or a novel technology developed for multiparametric flow imaging of a heterogeneous population of cells and sub-cellular elements using morphologic features (ImageStreamTM). Identification of fetal apoptotic bodies will be based on detection of cellular specific markers for apoptosis and trophoblasts followed by real-time PCR to detect fetal specific sequences (i.e. SRY gene locus) 2) To develop a model culture system to induce apoptosis, for studying fundamental properties associated with formation, stability and recovery of apoptotic bodies and apoptotic DNA. 3) To identify the quantity and molecular form(s) in which fetal DNA exists in maternal plasma. A combination of approaches will be used to characterize and quantify fetal DNA on the basis of size, existence of single or double stranded motives, stabilization by histones, and DNA methylation. Better understanding of the biology and molecular characteristics of fetal DNA in maternal circulation will enable improved methods for non-invasive prenatal genetic diagnosis and assessment of high risk pregnancies.